TotalExoRNA™ Kit (overall exosome immunocapture and RNA extraction kit) has been developed for the isolation and efficient extraction of high-quality total RNA (miRNA and mRNAs) from the overall exosome population from human biofluids or cell culture media. Exosome capture occurs on immunobeads precoated with exosome associated antibodies enabling overall exosome isolation. The immunobeads can capture the overall exosome population from a wide range of media, including cell culture supernatants and human biofluids (plasma, serum, urine, etc) as well as enrich some specific exosome subpopulations (tumor-derived exosomes). The captured exosomes are subsequently lysed with an optimized lysis buffer and total RNA is purified using spin columns with a fast and user-friendly process. Eluted RNA can be used for downstream analyses or stored at -80 °C. Exosome standards for positive control are also included in the kit. All our kits guarantee high specificity for exosomal RNA and high yield of total RNA (including small RNAs) than similar products.

Concentrate cell supernatant 10-20 fold in spin concentrator*. *The quantity of exosomes could vary between samples. A larger starting amount of sample should be used if the signal is weak.

Reagent preparation:

Bead Washing Buffer: Dilute bead washing buffer 5X to 1X with deionized water. Ensure there is no crystal precipitate. NOTE: If crystals are observed, dissolve them by warming up the concentrated 5X Washing Buffer bottle at 37 °C before proceeding with the dilution. Mix 5 mL of 5X Beads Washing Buffer with 20 mL deionized water for a final volume of 25 ml.

RNA Washing Buffer Solution: Add into the bottle containing RNA Washing Buffer the volume of pure ethanol (96 % ) indicated on the bottle's label to get the final ethanol concentration of approximately 70 %.

Elution Buffer and Lysis Buffer are ready to use.

Exosome binding:

Purified Exosomes (Lyophilized Standards): Purified exosomes do not require this binding step. If the samples are purified exosomes, skip to RNA extraction directly.

Unfractionated Samples:

Place 0.1 mL up to 1 mL of sample into low-binding tubes (not provided in the kit). Volumes suggested: 0.1 mL up to 0.5 mL for small RNA analysis, 0.5 mL up to 1 mL for mRNA analysis.

Add 1X PBS to the sample to get a final volume of 1 ml. (If you are using 1 mL of plasma, dilution is not necessary).

Add 10 μL of immunobeads.

Incubate sample-immunobead mixture overnight at 4 °C in a rotator.

Centrifuge at room temperature (RT) for 10 min at 5,000g.

Discard the supernatant.

Wash the beads:

Add 1 mL of Bead Washing Buffer.

Resuspend up and down 10-15 times.

Centrifuge at RT for 10 min at 5,000g.

Remove the supernatant being careful not to disturb the pellet.

Wash beads once again as indicated above.

RNA Extraction:

Lysis:

Purified Exosomes: Add 200 μL of Lysis buffer directly to lyophilized exosomes. Resuspend by pipetting and transfer to a fresh tube. Add 500 μL of Lysis buffer to reach a final volume of 700 μL. Incubate for 5 min at room temperature.

Unfractionated Samples: Add 700 μL of Lysis buffer directly on the bead pellet. Dissolve the pellet by pipetting up and down (beads must be totally dissolved). Incubate for 5 min at room temperature.

Elute the column with 15 μL of Elution buffer. Incubate 5 min at room temperature. Spin 2 min at 200g and 1 min at 14,000g. Keep the flow-through. Eluted RNA is now ready for downstream analysis or for storage at -80 °C

Sensitivity: TotalExoRNATM purified exosome RNA can be quantified and analyzed using the NanoDrop spectrophotometer (Thermo Scientific), although the measured concentration values are likely to end towards the bottom limit of detection of the instrument. For better quantification, we recommend the concomitant use of electropherogram-based technologies (e.g. Bioanalyzer, Agilent Technologies) or fluorimetric technologies (Qubit nano, Thermoscientific). Since most of the RNA contained in extracellular vesicles are small-non-coding RNAs (eg. miRNA), the expected Nanodrop profile, purity and yield are as shown in the representative Figure

TotalExoRNATM allows extraction of high quality of exosome-derived RNAs from low volumes of sample and better performance than competitors. Eﬃciency of TotalExoRNATM kit was tested vs a competitor kit for RNA extraction from healthy donor plasma derived exosomes.

Reagent Preparation

Immunobeads can be stored at +4 °C for up to 8 months.

The RNase free columns and elution tubes must be stored at room temperature.

All the opened buffers and diluted reagents including the bead washing buffer, RNA washing buffer, lysis buffer and the elution buffer should be stored at +4 °C.